Knowledge of the blood-brain barrier (BBB) has progressed rapidly over the past several years as new techniques (e.g. in vitro cell cultures) have become available. Improved technologies for the monitoring of barrier integrity in terms of electrical resistance and macromolecule permeability are readily accessible. Concomitant with the advancement of these techniques has come a wealth of knowledge regarding the relevant factors that promote the expression of a BBB phenotype particularly in endothelial cells.
The BBB maintains the homeostasis of the brain microenvironment, which is crucial for neuronal activity and function. Brain microvascular endothelial cells (EC) that constitute the BBB are responsible for the transport of metabolites, precursors and nutrients from the blood to the brain. The same cells are involved in the clearance of potassium and hydrogen ions from the brain. While blood-brain barrier EC retard the transcellular migration of most hydrophilic solutes, nutrients and sugars gain rapid access into the brain. In mammals and in higher vertebrates, the sites of the BBB are the complex tight junctions between EC that prevent the paracellular migration of hydrophilic molecules from blood to brain and vice versa. The perivascular glia process with encompass the basal lamina of the endothelial cells in the central nervous system influence the integrity of the tight junctions. Specialized transporters for sugars (e.g. glucose) and amino acids have been described in blood-brain barrier EC and account for the transendothelial permeability of otherwise membrane-impermeant substances.
As one may expect given the seemingly opposite properties of these BBB cells, two different subcellular mechanisms are responsible for the xe2x80x98barrierxe2x80x99 and xe2x80x98transportxe2x80x99 features of blood-brain barrier EC: tight junctions and specialized transcellular transporters, including micropinocytotic vesicles for macromolecules.
An often neglected aspect of the BBB relates to its capacity to act simultaneously as a barrier and as a transporter for any ion or molecule. For example, the BBB is virtually impermeant to intraluminal potassium (Kplasma,). However, brain (i.e. abluminal) potassium is transported to the blood by a specialized and topographically segregated Na/K-ATPase. Thus, by combining the tight junction-mediated xe2x80x98tightnessxe2x80x99 of the BBB with an asymmetric transporter, KCSF remains constant in spite of Kplasma variations or parenchymal increases resulting from neuronal activity.
Most of these specialized properties (tight junctions, micropinocytotic vesicles, transporters, ion homeostasis mechanisms) are bestowed on endothelial cells by the brain tissue. Peripheral capillaries that vascularize brain tissue acquire BBB properties. However, isolated blood-brain barrier EC lose their properties after culturing in vitro.
Therefore, two key factors must be present in order for central nervous system endothelial cells to express a barrier phenotype in vitro which distinguishes them from their peripheral counterparts. First, the exposure of the apical membrane to shear stress, which is generated by the flow of blood across the apical surfaces of the endothelial cells, is vital to promote growth inhibition and differentiation of endothelial cells. Also, the exposure to shear stress serves to induce metabolic changes that limit the oxygen and substrate consumption of such cells and allow for trafficking of metabolic fuels to the brain. A by-product of the metabolic changes induced by flow is an improved capacity for endothelial cells to handle oxidative stress. The second vital factor for BBB formation by endothelial cells is exposure of these cells to as yet unidentified xe2x80x9cpermissivexe2x80x9d or xe2x80x9cpromotingxe2x80x9d factors presumably secreted by glia, specifically astrocytes. The basis for this comes from several series of experiments documenting close apposition of astrocyte foot processes to endothelial cells in instances of barrier expression and the absence of such expression when astrocytes or astrocytic factors are lacking. Astrocytic influences promote both a variety of changes in gene expression in endothelial cells as well as phenotypic changes including segregation of transporters and enzymes.
The goal of any study of BBB physiology or biology is to reproduce as many aspects as possible of the in vivo endothelial cells. The apposition of endothelial cell and astrocyte cell cultures in physically separate, but biochemically contiguous compartments and the exposure of the endothelial cells to apical shear stress are primary features of any dynamic model of the blood-brain barrier. Furthermore, an in vitro BBB model should simulate as many of the following properties as possible: (1) expression of tight junctions between ECs and the relative lack of pinocytotic vesicles (commonly assessed by measuring trans-endothelial electrical resistance (TEER) or permeability to radioactive molecules of poor or negligible permeation such as sucrose or mannitol); (2) selective (and asymmetric) permeability to physiologically relevant ions, such as Na+ or K+; (3) selective permeability to molecules, based on their molecular weight and oil/water partition coefficient; (4) expression of BBB-specific transporters for metabolic substrates or building blocks necessary for neuronal and glial cell physiology; and (5) functional expression of mechanisms of active extrusion of otherwise permeable substances (such as antineoplastic agents).
A first attempt at an in vitro BBB model included the use of cone and plate viscometers as well as parallel plate apparatuses combined with semipermeable membranes that are able to generate shear stress and co-culture conditions. However, these models did not possess the three-dimensional architecture characteristic of brain tissue in situ and lacked the necessary glial factors. Another model design known in the art uses a hollow fiber apparatus to conduct BBB studies. This model results from a modification of a traditional cell culture system that is normally used for extensive culturing of non-EC cells. The general design of the hollow fiber apparatus is derived from attempts to develop a xe2x80x98cell factoryxe2x80x99. U.S. Pat. No. 3,821,087 to Knazek et al. and U.S. Pat. No. 4,220,725 to Knazek et al. describe cell culturing devices using hollow fibers. Since then, these cell culturing devices have been extensively exploited for mass production of rare cell types, antibody production, and modeling of organ-like structures such as the BBB. Ott et al. used a hollow fiber cell culture apparatus for studies of flow-mediated effects on endothelial cell growth. A cell culturing device that is commercially available is CELLMAX(copyright) from Spectrum Laboratories.
Applicant has co-authored several publications describing attempts to simulate the blood brain barrier utilizing cell culture models by co-culturing endothelial cells intraluminally (i.e., intracapillary) and glia extraluminally (i.e., extracapillary). These publications include xe2x80x9cA New Model of the Blood Brain Barrier: Co-Culture of Neuronal, Endothelial, and Glial Cells Under Dynamic Conditions,xe2x80x9d NeuroReport, Vol. 10, No. 1816, December 1999; xe2x80x9cUnderstanding the Physiology of the Blood Brain Barrier: In Vitro Models,xe2x80x9d News in Physiological Sciences, Volume 13, December 1998; xe2x80x9cDynamic In Vitro Modeling of the Blood Brain Barrier: A Novel Tool for Studies of Drug Delivery to the Brain,xe2x80x9d PSTT, Vol. 2, No. 1, January 1999; Morphological and Functional Characterization of an In Vitro Blood-Brain Barrier Model,xe2x80x9d Brain Research, No. 771, 1997; and Mechanisms of Glucose Transport at the BBB: An In Vitro Study, Brain Research 409, 2001 which are all hereby incorporated by reference in their entireties to the extent they discuss the utilization of cell culturing models to simulate the BBB.
However, although cell culture models may be used to model the BBB, the cell culture models known in the art have proven to be of limited applicability. For example, these models provide poor visualization of the intracapillary or extracapillary space to assess morphologic and/or phenotypic changes in the cells of interest, and do not appropriately provide the necessary access for the introduction of outside agents or samples. The volume of and physical access to the extracapillary space in these cell culture models known in the art allow for only cell suspension introduction. Finally, the cell inoculation volume and media requirements of these models are relatively large in light of the fact that these models are not reusable.
It is desirable to develop a cell and tissue culture modeling device and apparatus that addresses these limitations, but also adds functionality, modularity, and expandability to the experimental repertoire unavailable under the current configuration.
Generally, the present invention is directed to a cell and tissue culture modeling device and apparatus and method of using the same.
In one embodiment of the present invention, the cell and tissue culture modeling device includes a design amenable to light and fluorescence microscopy. For instance, the thickness of the device is within the resolution range of light and fluorescence microscopes, thus examination of the extracapillary space is achievable. The capacity to remove a single or multiple hollow fibers during the course of an experiment addresses the issue of visualization of the endothelial cell provides an advantage to the present invention. Also, the relative flatness of the device makes it modular and thus automation of simultaneous permeability determinations of compounds and multiplexing is possible.
In a preferred embodiment of the present invention, the device includes housing wherein at least a portion of the housing is removable to access the extracapillary space. This access to the extracapillary space provides for the introduction of tissue samples, neurochips, or other testing devices into the extracapillary space.
In a preferred embodiment of the present invention, the relative size of the extracapillary space to the intracapillary space is increased to accommodate the tissue samples in addition to cell suspensions. Adequate permeability measurements are still obtainable given the increase in the size of the extracapillary compartment. Also, additional access is provided to the extracapillary space through the use of access ports that have the ability to measure transendothelial electrical resistance.
In yet another alternative embodiment of the present invention, each component of the device is downsized to varying degrees such that the amount of cells required to initiate an experiment has been substantially reduced (by nearly four times) and the media volume required to perfuse the system and maintain cell viability (approximately half). When considering the use of expensive growth factors or the need to dispose of radiolabeled macromolecules, total media requirements can be an important issue.
Cell and tissue culture modeling devices have a variety of applications ranging from the in vitro modeling of the blood-brain barrier for focused individual study to fully automated microdialysis-driven permeability determinations for multiple biologically active molecules on a mass scale. Other relevant applications of the present invention include a clinically predictive tool for the efficacy of chemotherapeutic agents in the treatment of primary central nervous system malignancy, the incorporation of a brain slice to the system to simultaneously evaluate drug penetration and efficacy, as well as time course driven monitoring of gene expression profiles of endothelial cells and brain parenchymal cells under conditions which promote cell maturation and differentiation.
According to the present invention, a cell and tissue culture modeling device (hereinafter referred to as xe2x80x9cdevicexe2x80x9d)comprises a housing having an interior chamber and an inlet port and an outlet port. Both the inlet and outlet ports are in fluid communication with the internal chamber. The device also includes a plurality of hollow fibers disposed within the interior chamber. The hollow fibers traverse the length of the housing between the inlet port and the outlet port. Each of the hollow fibers has an interior defined as an intracapillary space. The inlet port permits fluid to enter the housing, via the intracapillary space of each hollow fiber, and exit the housing through the outlet port. The hollow fibers occupy only a portion of the internal chamber. The unoccupied portion of the internal chamber is defined as an extracapillary space. A portion of the housing is removable to access the extracapillary space.
The housing includes a pair of opposing end walls and a pair of opposing side walls. In at least one of the pair of opposing side walls, at least one access port is provided in fluid communication with the extracapillary space. In one of the end walls of the housing, the inlet port is provided. In the other end wall of the housing, the outlet port is provided. The housing also includes a top wall wherein at least a section of said top wall defines a top panel. The top panel is the portion of the housing that is removable to access the extracapillary space and is removably attached to the housing. The device may also include a gasket installed in between the top panel and the housing to create a watertight extracapillary space. The housing also includes a bottom wall wherein at least a portion of the bottom wall defines a bottom panel made of laboratory quality glass.
Each of the plurality of hollow fibers has a wall that includes a plurality of pores that provide fluid communication between the intracapillary space and the extracapillary space. The size of each of the plurality of pores is between about 0.01 xcexcm and about 0.50 xcexcm. The hollow fibers are formed of a material selected from the group consisting of polypropylene, polyester, polystyrene, polycarbonate, nitrocellulose compound, polyethylene, polysolfone, cellulose, polymethyl methacrylate, polyacrylonitrile, and polyvinylidene fluoride. The device of claim 6, wherein said plurality of hollow fibers are suspended and fixed in said inlet port and outlet port using an epoxy adhesive to create a watertight extracapillary space.
In another embodiment, the present invention provides a dynamic three-dimensional cell and tissue culture modeling apparatus (hereinafter referred to as xe2x80x9capparatusxe2x80x9d). The apparatus comprises at least one cell and tissue culture modeling device according to the present invention, a pump system, a media reservoir, and a first, second, and third conduit. The first conduit interconnects the media reservoir to the pump system, while the second conduit interconnects the pump system to the inlet port of each device, and the third conduit interconnects the outlet port of each device to the media reservoir. The second conduit is in fluid communication with the intracapillary spaces of the plurality of hollow fibers in the inlet port. The third conduit is in fluid communication with the intracapillary spaces of the plurality of hollow fibers in the outlet port. Preferably, the pump system is a variable speed pump system that generates pulsatile flow. Preferably, the first, second, and third conduit is gas permeable tubing. The apparatus may further comprise a first valve positioned between the pump system and the media reservoir and a second valve positioned between the pump system and each device to ensure unidirectional flow.
In another embodiment, at least one intracapillary space of the plurality of hollow fibers is inoculated with a first cell suspension. Preferably, the first cell suspension includes endothelial cells.
In yet another embodiment, the extracapillary space is inoculated with a second cell suspension. Preferably, the second cell suspension includes glial cells such as astrocytes.
Also, the present invention provides for a method of determining the permeability of an agent across a capillary wall comprising the steps of providing a cell culture model having a plurality of capillaries disposed within an interior chamber which defines an extracapillary space unoccupied by the plurality of capillaries, each of the plurality of capillaries including a plurality of pores that provide fluid communication between an intracapillary space and the extracapillary space; passing an agent having a known concentration through the plurality of intracapillary spaces; sampling the extracapillary space to provide an extracapillary space sample; and analyzing the extracapillary space sample to determine the permeability of the agent across each of the capillary walls. The plurality of intracapillary spaces may be inoculated with endothelial cells and the extracapillary space may be inoculated with glial cells such as astrocytes. A microdialysis-driven sample probe can accomplish the sampling step. A second cell culture model may be introduced to allow for the simultaneous determination of permeability values of at least two agents in a single experiment.
Furthermore, the present invention provides for a method of determining the efficacy of a drug comprising the steps of providing a model that exhibits the properties of a functional blood brain barrier, the model having a plurality of intracapillary spaces and an extracapillary space accessible by an access panel; placing a tissue sample into the extracapillary space; passing an agent through the plurality of intracapillary spaces; and analyzing the tissue sample for responsiveness to the agent. The tissue sample may be a cancerous tissue sample or a brain tissue sample. The agent that passes through the plurality of intracapillary spaces may be a chemotherapeutic agent. The method may further comprise the step of placing a neurochip in the extracapillary space before placing the brain tissue sample into the extracapillary space, wherein the brain tissue sample is placed onto the surface of the neurochip. The neurochip is capable of studying the electrophysiological activity of the brain tissue sample. In this scenario, the brain tissue sample may be an epileptic brain tissue sample and the agent may be an anticonvulsant agent. Finally, the method may further comprise the step of examining the tissue sample in the extracapillary space with a microscope.
Additionally, the present invention provides for a method of determining gene expression over time in cells comprising the steps of providing a cell culture model having a plurality of hollow fibers disposed within an interior chamber which defines an extracapillary space unoccupied by the plurality of hollow fibers, each of the plurality of hollow fibers includes an intracapillary space inoculated by a cell suspension; passing an agent through the plurality of intracapillary spaces; sampling at least one of the plurality of intracapillary spaces by removing at least one of the plurality of hollow fibers over time; removing the cellular material from at least one of the plurality of hollow fibers; and analyzing the gene expression of the cellular material. The cellular material may include RNA, DNA, metabolites, or protein.